Glutathione plays a key role in cellular detoxification. The interactions are often catalyzed by the glutathione S-transferases. Propylthiouracil has been found to competitively inhibit the glutathione binding site of these enzymes. Therefore, studies are being done using propylthiouracil as a specific in vivo probe and inhibitor of the glutathione binding site. In rats, large doses of PTU (150 mg/kg ip) inhibited the depletion of hepatic glutathione by azathioprine (a substrate for the transferases) and the storage of sulfobromophthalein and its excretion as a conjugate in bile. We also found that propylthiouracil could act like glutathione in protecting against covalent binding of acetominophen-reactive metabolite in vitro in a microsomal system and in vivo by a direct effect when given 30 minutes before acetaminophen. Preliminary evidence suggest that the mechanism for this direct protective effect is the formation of an adduct of propylthiouracil and the acetaminophen metabolite.